1. Field of the Invention
This invention relates to a method for the selective destruction of malignant cells in mammals based on metabolic differences between those cells and non-malignant (i.e., "normal") cells. More specifically, it relates to starvation of malignant cells which lack the enzyme necessary to convert methylthioadenosine to methionine by degrading plasma methionine and homocysteine.
2. History of the Invention
The amino acid methionine (MET) is necessary for the growth of normal and malignant cells. In certain malignant cells this requirement is absolute, i.e., without an adequate supply of MET, the cells die.
In mammalian cells, MET is obtained from three sources. It can be obtained in the diet, or through biochemical synthesis of MET from L-homocysteine (homocysteine) or methythioadenosine (MTA) (a product of the polyamine biosynthetic pathway). In the latter case, MTA is converted to MET by methyethioadenosine phosphorylase (MTAse).
In the past decade, researchers have identified many malignant cell lines which lack MTAse and cannot, therefore, convert MTA to MET. For example, Katamari, et al. , Proc. Nat'l Acad. Sci. USA, 78:1219-1223 (1981) reported that 23% of 3 human malignant tumor cell lines lacked detectable MTAse, while MTAse activity was present in each of 16 non-malignant cell lines studied. MTAse negative cells principally fulfill their requirement for MET through conversion of homocysteine. However, when homocysteine is not available, the cells will generally die.
L-methionine-L-deamino-y-mercaptomethane lyase (ED 4.4.1.11; METase) is known to degrade not only MET but also homocysteine. Theoretically, therefore, one could starve malignant cells which lack MTAse (i.e., MTAse negative cells) by degrading plasma MET and homocysteine with METase. Normal MTAse positive cells would be expected to fulfill their requirement for MET by the continued conversion of MTA to MET.
A rudimentary version of this approach was first proposed in 1972 by Kreis in Cancer Treat. Rprts., 63:1069-1072 (1972). Using 11 malignant cell lines in MET-free cultures, Kreis was able to inhibit the growth of certain of the malignant cells by applying METase to the cultures. Kreis also observed that 2 normal cell lines were partly "rescued" from the effects of MET starvation when homocysteine was added to the cultures. However, while these in vitro studies were encouraging, several obstacles were described by Kreis as being in the way of a successful in vivo use of METase in chemotherapy, including the unavailability of means to ensure the survival of normal cells in vivo, the potential immunogenicity of purified or partially purified enzyme, and the need for the enzyme to be resistant to degradation by proteolytic enzymes in vivo (Kries, Chemotherapy (Muggia, FM, ed., The Hague, Boston, and London: Martinus-Nijihoff, 1983), pp. 219-248).
Another obstacle to the development of a successful approach to MET starvation of malignant cells has been the need to identify which malignancies are suitable targets for the therapy; i.e., which malignancies are MTAse negative. To that end, an assay was developed which predicts whether a malignancy is MTAse negative by determining whether any catalytic activity is present in a cell culture (Seidenfeld, et al., Biochem. Biophys. Res. Commun., 95:1861-1866, 1980). However, because of the commercial unavailability of the radiochemical substrate required for the activity assay, its use in routine evaluations is not presently feasible. Moreover, the activity assay does not account for the catalytic lability of MTAse in vitro by detecting whether any of the enzyme is present in the cell culture regardless of whether it is catalytically active at the time that the assay is performed.
This limitation of the activity assay could be avoided by the development of an immunoassay which is sufficiently sensitive to detect relatively minute quantities of enzyme. However, the purification of the MTAse enzyme from natural sources to develop antibodies for use in immunological detection of MTAse has proven to be a laborious process which produces relatively poor yields (Raglone, et al., J. Biol. Chem., 261:12324-12329, 1986).
Even if adequate means were developed to detect MTAse negative cells, production of an adequate supply of METase from natural sources has been as difficult as the production of MTAse. Production of METase by means other than purification of the native enzyme has not yet been achieved, in part because the gene for METase has (to date) been only partially sequenced (Nakayama, et al., Biochem, 27:1587-1591, 1988).
For all of these reasons, an effective approach to in vivo MET starvation of MTAse malignant cells has remained elusive. The present invention addresses this need.